The present invention relates to sensitive lateral-flow methods and devices for determining the presence and/or amount of small and large analytes in fluid samples. The present invention provides a direct or positive detection result (i.e. increasing signal with increasing analyte concentration) in a sequential binding format.
Analytical tests have been developed for the routine identification or monitoring of physiological and pathological conditions (e.g., pregnancy, cancer, endocrine disorders, infectious diseases) using different biological samples (e.g., urine, serum, plasma, blood, saliva), and for analysis of environmental samples (e.g., natural fluids and industrial plant effluents) for instance for contamination. Many of these tests are based on the highly specific interactions between specific binding pairs. Examples of such binding pairs include antigen/antibody, hapten/antibody, lectin/carbohydrate, apoprotein/cofactor and biotin/(strept)avidin. Furthermore, many of these tests involve devices (e.g., solid phase, lateral-flow test strips, flowthrough tests) with one or more of the members of a binding pair attached to a mobile or immobile solid phase material such as latex beads, glass fibers, glass beads, cellulose strips or nitrocellulose membranes (U.S. Pat. Nos. 4,703,017; 4,743,560; 5,073,484).
Immunochromatographic assays fall into two principal categories: xe2x80x9csandwichxe2x80x9d and xe2x80x9ccompetitive.xe2x80x9d In general, sandwich immunochromatographic procedures call for mixing the sample that may contain the analyte to be assayed with antibodies to the analyte. These antibodies are mobile and typically are linked to a label or another signaling reagent, such as dyed latex, a colloidal metal sol, or a radioisotope. This mixture is then applied to a chromatographic medium containing a band or zone of immobilized antibodies to the analyte of interest. The chromatographic medium often is in the form of a strip that resembles a dipstick. When the complex of the molecule to be assayed and the labeled antibody reaches the zone of the immobilized antibodies on the chromatographic medium, binding occurs and the bound, labeled antibodies are localized at the zone. This indicates the presence of the molecule to be assayed. This technique can be used to obtain quantitative or semi-quantitative results. Examples of sandwich immunoassays performed on test strips are described in U.S. Pat. Nos. 4,168,146 and 4,366,241, each of which is incorporated herein by reference.
In competitive immunoassays, the label is typically a labeled analyte or analyte analogue that competes with any unlabeled analyte present in the sample for binding to an antibody. In such competitive assays, the analyte and labeled tracer molecule are simultaneously introduced to the binding agent such that these molecules compete for binding sites. Competitive immunoassays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are those disclosed by U.S. Pat. Nos. 4,235,601, 4,442,204 and 5,208,535, each of which is incorporated herein by reference.
Solid phase immunoassay devices, whether sandwich- or competition-type, provide sensitive detection of an analyte in a biological fluid sample. Solid phase immunoassay devices incorporate a solid support to which one member of a ligand-receptor pair, usually an antibody, antigen, or hapten, is bound. Common early forms of solid supports were plates, tubes, or beads of polystyrene, which were known from the fields of radioimmunoassay and enzyme immunoassay. More recently, a number of porous materials such as nylon, nitrocellulose, cellulose acetate, glass fibers, and other porous polymers have been employed as solid supports.
In the more common forms of dipstick assays, as typified by home pregnancy and ovulation detection kits, immunochemical components such as antibodies are bound to a solid phase. The assay device is xe2x80x9cdippedxe2x80x9d for incubation into a sample suspected of containing the subject analyte. Enzyme-labeled antibody is then added, either simultaneously or after an incubation period. The device next is washed and then inserted into a second solution containing a substrate for the enzyme. The enzyme-label, if present, interacts with the substrate, causing the formation of colored products, which either deposit as a precipitate onto the solid phase or produce a visible color change in the substrate solution. EP-A 0 125 118 discloses such a sandwich type dipstick immunoassay. EP-A 0 282 192 discloses a dipstick device for use in competition type assays.
Flow-through type immunoassay devices (such as test strips) were designed to obviate the need for incubation and washing steps associated with dipstick assays. U.S. Pat. No. 4,632,901 discloses a sandwich immunoassay device wherein antibody (specific to a target antigen analyte) is bound to a porous membrane or filter to which a liquid sample is added. As the liquid flows through the membrane, target analyte binds to the antibody. The addition of sample is followed by addition of labeled antibody. The visual detection of labeled antibody provides an indication of the presence of target antigen analyte in the sample.
Migration assay devices usually incorporate within them reagents that have been attached to colored labels, thereby permitting visible detection of the assay results without addition of further substances. See, for example, U.S. Pat. No. 4,770,853; WO 88/08534; and EP-A 0 299 428.
There are a number of commercially available lateral-flow type tests and patents disclosing methods for the detection of large analytes (MW greater than 1,000 Daltons). U.S. Pat. No. 5,229,073 describes a semiquantitative competitive immunoassay lateral flow method for measuring plasma lipoprotein levels. This method utilizes a plurality of capture zones or lines containing immobilized antibodies to bind both the labeled and free lipoprotein to give a semi-quantitative result.
U.S. Pat. No. 5,591,645 provides a chromatographic test strip with at least two portions. The first portion includes a movable tracer and the second portion includes an immobilized binder capable of binding to the analyte. Additional examples of lateral-flow tests for large analytes are disclosed in the following patent documents: U.S. Pat. Nos. 4,168,146; 4,366,241; 4,855,240; 4,861,711; 5,120,643; European Patent No. 0296724; WO 97/06439; and WO 98/36278.
There are also a limited number of lateral-flow type tests for the detection of small-analytes (MW 100-1,000 Daltons). Generally, these small analyte tests involve xe2x80x9ctypicalxe2x80x9d competitive inhibition to produce negative or indirect reporting results (i.e., reduction of signal with increasing analyte concentration), as exemplified by U.S. Pat. No. 4,703,017.
Several approaches have been developed for detecting small analytes using lateral-flow tests that produce positive or direct reporting results (i.e., increase in signal with increasing analyte concentration). These include, for instance, U.S. Pat. Nos. 5,451,504; 5,451,507; 5,798,273; and 6,001,658.
U.S. Pat. No. 5,451,504 provides a method with three specific zones (mobilization, trap and detection) each containing a different latex conjugate to yield a positive signal. The mobilization zone contains labeled antibody to bind the analyte in the sample. In the trap zone, unbound, labeled antibody is then trapped by immobilized analyte analog. The detection zone captures the labeled analyte-antibody complex. A disadvantage of this method is that the analyte-analog in the trap zone competes with the labeled analyte-antibody complex formed during migration and may cause false negative results.
U.S. Pat. No. 5,451,507 describes a two-zone, disconnected immunochromatographic method. The first zone has non-diffusively bound reagent that binds with a component, e.g., an analyte analog bound to, or capable of becoming bound to, a member of a signal producing system. The second zone binds to the component only when the analyte to be tested is present. The distance the component migrates into the second zone is directly related to the concentration of analyte.
U.S. Pat. No. 5,798,273 discloses a lateral flow device that includes a capture zone with immobilized analyte analog and one or more read-out zones to bind labeled analyte-analog. A disadvantage of this disclosed method is the requirement to premix sample, antibody and colored label prior to application to the sample addition area of the lateral flow device.
U.S. Pat. No. 6,001,658 discloses a test strip device with a diffusible, labeled binding partner that binds with analyte, an immobilized analyte, and a detection area containing an immobilized antibody.
A continuing need exists for a sensitive, rapid and single step method to detect and quantify both large and small analytes at low concentrations (such as ng/ml or less).
The present invention provides sensitive, rapid devices (in the form of a test strip) for determining the presence and/or amount (at ng/ml levels) of small and/or large analytes in a fluid sample. The invention also provides methods for the determination of the presence and/or amount of one or more components (e.g., analytes) in a sample. Results from the methods and devices disclosed herein can be positively read directly from the assay device by visual inspection or using an electronic reader (such as a scanner).
The methods and devices disclosed herein can be used to detect analytes in various types of fluid, including biological specimens (such as blood, serum, plasma, urine, saliva, milk) and environmental samples (such as industrial plant effluent or natural fluids). Any known analyte with an appropriate analyte-specific antibody or other binding partner can be easily detected and/or quantified using the disclosed methods and devices. In certain examples of embodiments, a tracer is used, such as colored or otherwise detectable particles (e.g., colored latex or colloidal gold) conjugated to the analyte or an analyte-analog, which tracers are collectively referred to as the conjugate.
Devices of the invention include a mobilization zone, a sample application area, and primary and secondary capture areas. Each of the capture areas includes an immobilized binding partner (such as an antibody) having a binding affinity for the analyte being tested and for the detectable conjugate. A mobilization zone, sample application area, and primary and secondary capture areas are in fluid continuous contact with each other, e.g., on a lateral flow chromatography strip. In these devices, the first immobilized binding partner binds the analyte and detectable conjugate. However, to the extent that analyte is present in the sample, the labeled conjugate is less able or unable to bind in the primary capture zone and instead continues to migrate along the strip toward the secondary capture zone, where it binds with the secondary binding partner. The labeled conjugate therefore provides a positive signal, in proportion to the amount of analyte present in the sample, in the secondary capture zone.
In some embodiments, the specific binding agent in the primary capture zone may have a higher affinity for the analyte than the labeled conjugate, such that the conjugate is less preferentially bound in the primary capture zone and preferentially passes through to the secondary capture zone. However, the binding agent in the primary capture zone may have an equal affinity for the analyte, and the labeled analyte will still pass through the primary capture zone to bind in the secondary capture zone and emit a signal proportional to the presence of analyte in the sample. It is also possible that the binding agent in the primary capture zone may have a lower affinity for the analyte in some embodiments, and still provide a substantially quantitative signal from the secondary capture zone, with the signal proportional to the amount of analyte in the sample.
Other embodiments of the test strip permit sequential migration of the analyte and labeled analyte analog, such that the analyte migrates in advance of the labeled conjugate, to be bound by the specific binding agent in the primary capture zone. Since sites in the primary capture zone are already occupied when the labeled conjugate reaches the primary capture zone, the conjugate continues to migrate along the strip to the secondary capture zone. The labeled conjugate then provides a signal in the secondary capture zone, which signal is proportionate to the amount of analyte in the sample.
Certain methods of this invention involve introducing a liquid sample (which is suspected of containing the analyte to be tested for) on to the test strip, and permitting the sample to migrate along the test strip by capillary action from the application area to and through the first and secondary binding zones. The tracer conjugate may be present in the application zone, in the path of migration, or applied separately to the strip. The conjugate also migrates along the test strip, for example slightly behind the sample, so that any analyte in the sample contacts the first binding partner before the analyte-tracer conjugate. If the analyte is present in the sample, it will bind to the first binding partner, and occupy binding sites that are then not available to bind the labeled conjugate. Hence the conjugate will migrate along the strip to the secondary binding zone, where the presence and/or intensity of the detectable signal positively indicates an amount of analyte in the sample.
The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying Figures.